The aim of this research is to investigate the nature and location of the topogenic information that is responsible for targeting proteins to peroxisomes of Candida topicalis. These studies should provide fundamental information about an intriguing problem in cell biology for which no molecular information is yet available. They may also shed light on several human diseases in which peroxisome assembly is defective. Peroxisomes are nearly ubiquitous in eukaryotic cells and have essential functions including fatty acid catabolism, gluconeogenesis, and plasmalogen biosynthesis. Peroxisomes resemble mitochondria and chloroplasts in that the organelles import posttranslationally proteins that are synthesized on free polyribosomes. Peroxisomes differ in that their proteins generally lack cleavable transit peptides, whereas most mitochondrial and chloroplast proteins are synthesized as larger precursors with topogenic aminoterminal peptides that are removed proteolytically upon import. Recently we have made a cDNA library and established an in vitro assay for import of proteins into peroxisomes. With these tools, we now plan to investigate the topogenic information in two peroxisomal proteins, an acyl-CoA oxidase and a 16 kDa polypeptide. Preliminary evidence indicates that sufficient information is present in the carboxyterminal 40% of acyl-CoA oxidase to direct its import into peroxisomes. The location of the targeting sequences within these genes will be determined by deletion analyses and fusions with dihydrofolate reductase. The exact nature of the topogenic sequences will be further investigated by site-directed mutagenesis.